The nucleus basalis magnocellularis (nBM) provides the primary source of cholinergic input to the cortex. Neonatal lesions of the nBM produce transient reductions in cholinergic markers, persistent abnormalities in cortical morphology, and spatial navigation impairments in adult mice. The present study examined sex differences in the effects of an electrolytic nBM lesion on postnatal day 1 (PND 1) in mice on behavior and neurochemistry in adulthood. Mice were lesioned on PND 1 and tested at 8 weeks of age on a battery of behavioral tests including passive avoidance, cued and spatial tasks in the Morris water maze, simple and delayed nonmatch to sample versions of an odor discrimination task, and locomotor activity measurements. Following behavioral testing, mice were sacrificed for either morphological assessment or neurochemical analysis of a cholinergic marker or catecholamines. There were no lesion or sex differences in acquisition or retention of passive avoidance, performance of the odor discrimination tasks, or activity levels. Control mice showed a robust sex difference in performance of the spatial water maze task. The lesion produced a slight cued but more dramatic spatial navigation deficit in the water maze which affected only the male mice. Neurochemical analyses revealed no lesion-induced changes in either choline acetyltransferase activity or levels of norepinephrine or serotonin at the time of testing. The subsequent report shows a sex difference in lesion-induced changes in cortical morphology which suggests that sexually dimorphic cholinergic influences on cortical development are responsible for the behavioral deficits seen in this study.